NREL's CelA Catalyzes Plant Cell Walls Faster

January 12, 2015

NREL Senior Scientist Roman Brunecky examines the molecular weight of the enzyme CelA
on a gel in the Protein Chemistry Lab in the Field Test Laboratory Building on NREL's
Golden, Colorado, campus.Photo by Dennis Schroeder

Scientists at the Energy Department's National Renewable Energy Laboratory (NREL)
have developed an enzyme that could change the economics of biofuel conversion by
converting biomass to sugars up to 14 times faster and much cheaper than competing
catalysts in enzyme cocktails today.

This enzyme is called CelA, a cellulase from the bacterium Caldicellulosiruptor bescii, and the fact that it's from a bacterium, and not a fungus, is just one reason why
it is such a potential game-changer. Here are some others:

Unlike most catalysts, CelA can digest not one, but two major components in biomass:
both cellulose and xylan.

CelA works in two mechanical realms, not just one. It is an ablater, scraping the
valuable material off the cell walls of the plants. But it is also a borer, digging
deep into the wall to grab more of the digestible biomass. It is the only enzyme known
to dig pits into biomass; others only ablate.

It can operate at much higher temperatures than other enzymes. That's important because
high temperatures mean faster action. Also, because it can operate above the boiling
point of alcohol, the alcohol is separated naturally, saving a costly step in the
conversion process—and the high temperatures kill many of the microorganisms that
would otherwise interfere with the process.

In one scenario, the best commercially used enzyme converted sugars at a 30% extent
in seven days. CelA converted to double that extent. And while it took the alternative
enzyme seven days to achieve that conversion, CelA, with a small boost from an extra
beta glucosidase, achieved double in just about two days.

"If you can achieve in one day what typically takes seven, you are saving the better
part of a week of processing," NREL Senior Scientist Roman Brunecky said. "And that
can have a huge economic impact."

Just CelA alone is four to five times faster at breaking down sugars than the enzymes
in today's cocktails. A more typical usage would be CelA combined with a beta glucosidase—the
improvement that makes it 14 times faster.

The remarkable discovery comes at a time when the dogma in the industry was that the
important discoveries already had been made: that another cellulase, Cel7A, was the
key enzyme to be the backbone of all the cocktails. NREL's discovery not only reveals
that there is an intriguing enzyme that can work several times faster, but it is also
a reminder that nature still has secrets to uncover.

If the enzyme continues to perform well in larger tests, it could help drive down
the price of converting cellulose and, with it, the price of everything from jet fuel
to ethanol, butanol, drop-in fuels, and numerous chemicals.

NREL has filed for patent protection on the enzyme formulation and the improvements
made to the unusual enzyme.

CelA Both Scrapes and Digs, Deconstructing Biomass Faster

Conventional cellulases such as Cel7A (left side of figure) use a surface ablation
strategy to deconstruct cellulose, converting single layers of cellulose strands.
CelA (right side) utilizes both this surface ablation strategy as well as an entirely
new pit-formation mechanism to excavate down into the cellulose strands.Image by Bryan Donohoe, NREL

Lignocellulosic biomass is the most plentiful and sustainable resource on Earth, largely
made up of plant residuals that would otherwise go unused and left to decay. Using
this biomass as a source of alternative fuels can help offset the world's dependence
on fossil fuels and reduce greenhouse gas emissions. Using electron microscopy, the
NREL researchers and their partners at the University of Georgia found that CelA not
only ablates the cell wall of lignocellulosic biomass, but excavates cavities into
the surface. CelA also worked faster on raw biomass than it did on biomass pre-treated
with chemicals.

The discovery was the unexpected result of very thorough imaging and analysis by Bryon
Donohoe of NREL's Biomass Surface Characterization Laboratory research team.

The researchers found that the size of the holes in the plant walls was about the
same size as the enzymes themselves. The inability of the enzyme to digest a space
on the surface that is any bigger than its own size is probably the reason its digs
holes deep into the stuff it is digesting. Scraping and digging gets the job done
faster than just employing a single approach.

If CelA proves reliable as it is scaled up to manufacturing levels, it could mean
lower fuel prices in 2022, when the Energy Independence and Security Act mandates
annual production of 36 billion gallons of fuel made from the non-food part of plants.

Biomass is composed of three types of polymers that are intermeshed to form plant
cell walls: cellulose, xylan, and lignin. Each of the three polymers typically requires
several types of enzymes to deconstruct them to soluble species that can then be upgraded
to ethanol, drop-in fuels, or chemicals.

But CelA offered another surprise: an appetite for not just cellulose but xylan, too.
Xylan, which wraps around the cellulose fiber, can contribute significantly to the
manufacture of biofuels if its stubborn sugars can be released. "That was absolutely
a novel feature that no one knew about—CelA's ability to break down xylan," Brunecky
said.

First Found in Warm-Water Pools in Russia

CelA is a potential game-changer because it combines the activities of all four of
the major enzyme types, as illustrated here.Image by Alex Berlin, Novozymes, Inc.

C. bescii, producer of the remarkable CelA enzyme, was first discovered in warm-water pools
in the Valley of Geysers on Russia's Kamchatka Peninsula in the 1990s. Russian scientists
found the enzyme somewhat promising, but it wasn't until the NREL researchers conducted
a thorough analysis and added improvements that its remarkable potential was realized.

NREL found some surprising properties. For example, even though CelA contains naturally
occurring endoglucanase, the researchers found that adding more of the substance greatly
accelerated the rate at which it broke down sugars.

CelA has its own beta-glucosidase activity, but NREL researchers checked to see how
it would perform if more of the beta-glucosidase was added. That's when the rate really
accelerated.

"You'd think that nature would already have evolved an optimal mix in a single enzyme,"
said Brunecky, lead author of the Science paper. "You expect to see maybe a 20% to 30% improvement when you add a beta glucosidase
to a cocktail—not the doubling or tripling of the rate of conversion and the increase
of rates by a factor of 10 that we got with CelA. Nobody expected the improvement
to be this high."

"The bacteria that secrete the promising CelA thrive in temperatures of 80°C to 90°C,
close to boiling," said NREL Senior Scientist Yannick Bomble, who is the senior author
of the Science paper. "That's an advantage because the early pre-treatment process requires temperatures
greater than 100°C to remove unwanted materials. The next step requires cooling the
temperature to the preferred range of the enzyme. In the case of CelA, the temperature
doesn't have to drop as much—another way to save money."

In a letter of comment in the same issue of Science, Alex Berlin of biotechnology company Novozymes noted that CelA's ability to operate
at high temperatures "would be seen by many in the biomass biorefinery industry as
an advantage" because it "dramatically reduces the chances of bacterial contamination"
while lowering the viscosity of the mixtures.

The BioEnergy Science Center, one of three Bioenergy Research Centers supported by the Office of Biological and
Environmental Research in the Energy Department's Office of Science, provided the
funding for the research. Lead author of the paper was NREL scientist Roman Brunecky.
Others from NREL include Markus Alahuhta, Bryon S. Donohoe, Michael F. Crowley, Michael
G. Resch, Vladimir V. Lunin, Michael E. Himmel, and corresponding author Yannick J.
Bomble. Co-authors from the University of Georgia include Irina A. Kataeva, Sung-Jae
Yang, and Michael W.W. Adams.

Finding Indicates a New Third Way to Degrade Plant Cell Walls

A blue-stained image of protein separated from a CelA enzyme gives scientists a better
idea of the enzyme's properties and potential.
Photo by Dennis Schroeder

Before the discovery of CelA's remarkable properties, scientists knew of two ways
that enzymes go about degrading plant cell walls. One is to secrete free, complementary
enzymes that hydrolyze cellulose, and the other is to use large enzymatic assemblies,
called cellulosomes, to assemble scaffolds to attack the walls. The new finding indicates
that there is a third way.

"CelA is the most efficient single cellulase we've ever studied, by a large margin,"
Bomble said. "It is an amazingly complex enzyme, combining two catalytic domains with
three binding modules. The fact that it has two complementary catalytic domains working
in concert most likely makes it such a good cellulose degrader."

The NREL researchers led by Michael Himmel aren't resting on their laurels, or their
patents. They're examining the other enzymes secreted by the organism. They're also
using what they've learned from CelA to help improve cellulase enzymes that are more
compatible with the enzyme formulations used today.

Next Step: Boost Enzyme Yields

The next step toward using CelA at commercial scale is to express large amounts of
the enzyme in existing production systems, and to boost enzyme yields from the native
organism, C. bescii. Most of the large enzyme companies have the expertise to do that, so commercialization
looks like a good bet.

"We are learning a lot about the evolution of these cellulases, how they can thrive
in extreme environments, and how they operate on biomass," Brunecky said.

"This discovery could reshape the landscape of commercial cellulase cocktail design,"
said Paul Gilna, director of the BioEnergy Science Center.